Downloaded from orbit.dtu.dk on: Nov 08, 2017 Metabolomics – an analytical strategy for identification of toxic mechanism of action Skov, Kasper; Hadrup, Niels; Smedsgaard, Jørn; Frandsen, Henrik Lauritz Publication date: 2015 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Skov, K., Hadrup, N., Smedsgaard, J., & Frandsen, H. L. (2015). Metabolomics – an analytical strategy for identification of toxic mechanism of action. Søborg: National Food Institute, Technical University of Denmark. 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Metabolomics – An Analytical Strategy for Kasper Skov PhD Thesis 2015 Summary Humans are exposed to chemicals from diverse sources such as foods, pharmaceuticals, cosmetics and the air (Monosson 2005), which may affect human health, even causing serious disease or death (Nielsen et al 2010a). Toxicology is concerned with the study of toxic effects exerted by chemicals on a living organism, but also associated to issues related to poisons, being it clinical, industrial, or legal. Metabolism is the set of chemical reactions that allow an organism to maintain its functions, whereas metabolites are its intermediates and products. The complete set of small molecule metabolites is referred to as the metabolome and the comprehensive and quantitative analysis of all metabolites is denoted metabolomics (Fiehn 2001). The effects of toxic compounds as physiological or chemical induced changes in the mammalian body are reflected in the plasma metabolome. In toxicology, compounds with an effect on hormone regulation have attracted much attention. These are the so‐called endocrine disrupters which mimic natural endogenous hormones and are suspected to be involved in the observed decreased fertility in Denmark over the last two decades. Since endocrine disrupting chemicals (EDCs) have effects on humans at low concentration, one of the present challenges in toxicology is to develop methods capable of measuring exposures and effects at the low end of the scale. The aim of the present work was to scrutinize the impact toxic chemicals can have on the metabolome, in particular EDCs at low, human relevant concentrations. In order to accomplish this, a sample preparation technique suited to handle a wide range of metabolites in the plasma metabolome was developed. The technique included sub‐fractionation by solid phase extraction into three sub‐samples. The resulting method showed approximately 2.5 times more molecular features compared to that obtained if only protein precipitation was applied. The performance of the method was investigated using plasma samples from rats administered to the environmental pollutant perfluorononanoic acid (PFNA) and was applied in the metabolome analyses throughout the thesis. Humans are simultaneously exposed to multiple chemicals, many of which can be detected in human body fluids; however, the consequences of low dose exposure to complex mixtures of chemicals are poorly understood. By use of two omics approaches, metabolomics and transcriptomics, the effects on rats caused by exposure to a 14‐compound mixture (Mix) ± PFNA were profiled. The applied technologies provided complementary information allowing for a detailed analysis of the affected signalling pathways. Mix alone caused reduced lipid concentration evident in plasma. The hepatic effects on lipid metabolism were mainly driven by PFNA by activation of the PPAR receptors. This study verifies that a chemical mixture given at 1 high‐end human exposure levels can affect lipid homeostasis. In a follow‐up study on the importance of exposure to complex real‐world mixtures, data suggested that mixtures of environmental chemicals at doses approaching high‐end human exposure levels can cause a hormonal imbalance, with increased plasma corticosterone levels, and disturb steroid hormones and their regulation. To evaluate effects caused by low‐dose exposure, data from the PFNA study was used alongside data from a bisphenol A study. Plasma from pregnant rats and their offspring exposed to bisphenol A at an exposure concentration of 25 and 250 µg/kg bw/day were analyzed. A decrease in monoacylglycerol(18:0) and monoacylglycerol(16:0) in the mother animals was observed, while for the male offspring, increase in lyso‐ phosphotidylcholine plasma concentration was observed. The developed platform detects three main groups of metabolites; a phospholipid fraction, a lipid fraction and a polar fraction and reveals changes in the metabolome that could not be foreseen using regular toxicological approaches. The study presents a new approach which improves the basic biochemical understanding of toxic exposures to the rat. From this data it is feasible not only to determine the endpoint of the toxic exposure, but to suggest biochemical precursors to the exposure. 2 Resumé (In Danish) Mennesket er eksponeret for kemikalier fra mange forskellige kilder, blandt andet mad, medikamenter, kosmetik og atmosfærisk luft (Monosson 2005). Mange af disse kemikalier kan påvirke menneskets helbred, i nogle tilfælde påføre store skader og i værste fald slå folk ihjel (Nielsen et al 2010a). Toksikologien er den videnskab der beskæftiger sig med studiet af kemikaliers toksiske effekter på den levende organisme. Den er også associeret til spørgsmål relateret til giftstoffer, vedrørende kliniske, industrielle eller juridiske problemer. Metabolisme er det sæt af kemiske reaktioner som er essentielle for at opretholde organismens funktioner, mens metaboliter er dens intermediater og produkter. Den samlede mængde af metaboliter benævnes metabolomet og den omfattende og kvantitative analyse af alle metaboliter kaldes metabolomics (Fiehn 2001). Ændringer forårsaget af stoffer der fysiologisk eller kemisk påvirker den menneskelige krop kan observeres i plasma metabolomet. Stoffer, der påvirker hormonreguleringen, har fået stor opmærksomhed i toksikologien. Disse stoffer er såkaldte hormonforstyrrende stoffer, som efterligner de naturlige hormoner. I Danmark er der observeret en nedsat fertilitet over de sidste to årtier, og det menes at de hormonforstyrrende stoffer kan være medvirkende til denne nedgang. Eftersom de hormonforstyrrende stoffer har effekt på mennesket ved lav koncentration er en af de nuværende udfordringer i toksikologien, at udvikle metoder til måling af ændringer ved eksponering ved lave koncentrationer. Formålet med denne afhandling var at undersøge påvirkningen af toksiske kemikalier på metabolomet, især med fokus på de hormonforstyrrende stoffer ved lav human relevant koncentration. For at kunne opfylde dette formål, blev en procedure, som var baseret på en sub‐fraktionering udført ved hjælp af solid‐fase ekstraktion, udviklet til analyse af et bredt udsnit af metaboliter. Metoden kunne detektere 2,5 gange flere molekylære features end en metode der kun anvendte protein fældning, og blev testet i et rotteforsøg, hvor dyrene var eksponeret til et miljøkemikalium, perfluornonanone syre (PFNA). Denne fremgangsmåde blev anvendt i metabolom analyserne i hele denne afhandling. Mennesket er påvirket af mange kemikalier hver dag, hvoraf nogle kan blive detekteret i kropsvæsker. Men der er mangel på forståelse for, hvordan komplekse blandinger af disse stoffer vil påvirke os. For at undersøge dette brugte vi en dels metabolomics og dels transcriptomics tilgang for at undersøge effekten af PFNA med og uden en mix af 14 relevante kemikalier. Disse to fremgangsmåder blev brugt i kombination til at analysere en mulig påvirkning af biologiske pathways. Blandingen viste effekt på fedtstofskiftet 3 udtrykt ved en nedgang i plasma fedtstofferne, mens effekterne på leveren primært var fra PFNA ved aktivering af PPAR receptorerne. Studiet viste således at kemikalieeksponering, ved koncentrationer relevante for mennesker, kan påvirke fedtstofmetabolismen. Derudover viste forsøget at lav dosis kan påvirke hormonniveauerne i blodet hos mennesket, ved en forøget corticosterone plasma koncentration, samt reguleringen af disse. For yderligere at kunne evaluere lavdosiseksponering blev data fra PFNA studiet anvendt sammen med data fra et bisphenol A studie. Gravide rotter blev eksponeret til 25 og 250 µg/kg/dag, og blodet fra mødre og afkom blev analyseret. En nedgang i monoacylglycerol(16:0) og monoacylglycerol(18:0) blev observeret i mødrene, mens det mandlige afkom viste et øget niveau af lyso‐phosphocholine. Den udviklede analytiske platform detekterede således tre grupper af metaboliter; fosfolipider, lipider og en polær fraktion. Platformen viste ændringer som ikke ville være fundet ved regulære toksikologiske studier. Studierne i denne afhandling præsenterer en ny strategi til at evaluere biokemiske ændringer i rotter efter toksisk eksponering. Data fra dette studie viser, at det ikke kun er